MS response factors) were plotted as a function of the analyte concentrations (µg/kg). The correlation coefficients and regression equations were determined for each calibration curve.
5.2.5.6 Quality control samples. In order to verify the precision and accuracy of the analytical method, certified quality control samples [FAPAS (Cat no T22110QC) and Biopure (Cat no QCM3C2)] containing the mycotoxins at the expected concentrations ranges were included.
5.2.6 Correlation between Fusarium spp. occurrence and mycotoxin levels
Correlations were determined between the concentrations of the respective multiple mycotoxins and levels of F. verticillioides, F. graminearum, F. proliferatum and F. subglutinans in samples as determined with a validated quantitative Real-time PCR method (qPCR) (Chapter 4).
5.2.7 Statistical analyses
The NCSS Version 11 (NCSS, 2019) software was used for statistical analysis. Data was analysed within a generalised linear model ANOVA. P<0.05 was used as statistical significance. Correlation coefficients (R) were determined using R software, version 4.02 (Harris, 2018). The fungal genomic DNA values used in this exercise were obtained by using qPCR (Chapter 4).
antimicrobial agent to whole grains during storage (Oguntade and Adekunle, 2010). The raw grains are ground into flour and used to prepare porridge, confectionary, and alcoholic and non-alcoholic beverages. Production of malted sorghum and pearl millet involves cleaning, soaking in water (42-46% moisture content) and germination for 4-5 days, which produces solubilized nutrients and results in an increase in temperature (Agbor Asuk et al., 2020). Raw and processed sorghum and pearl millet samples were collected postharvest respectively from smallholder farmers in the Oshakati region, and from open markets in Oshakati and Ondangwa. The levels of multiple mycotoxins in samples were determined using a validated LC-MS/MS analytical technique.
The LC-MS/MS analytical specifications for each mycotoxin are summarised in Table 5.1. The results of the analytical validation experiments concerning sorghum and pearl millet are summarized in Tables 5.2 and 5.3, respectively. LC-MS/MS chromatograms of the respective analytical standards were separately compared with chromatograms and data of the blank sorghum and millet matrixes as well as selected samples. The results indicated that the blank sorghum and pearl millet contained no mycotoxins. Selectivity of the method was confirmed by the absence of co-eluting peaks (Figure 5.1). The percentage recoveries for the individual mycotoxins (68-95%) remained constant between runs. Results indicated that the extraction of FB1 and FB2 was more effective (P<0.05) from pearl millet than from sorghum. The LOQ levels for AFB1 (2 µg/kg), OTA (20 µg/kg), DON (100 µg/kg),ZEA (10 µg/kg), NIV (10 µg/kg) and MON (10 µg/kg) were the same for sorghum and pearl millet, while the LOQ levels for FB1, FB2 and FB3 (5-20 µg/kg) were higher in sorghum than in pearl millet (2-10 µg/kg). These differences could be attributed to the polarity of the extraction solvent in relation to the solubility of the mycotoxins in the grains. Replicate analysis of samples containing known amounts of the respective mycotoxins, resulted in means within 15% from the theoretical values, confirming the accuracy of the method. For increased accuracy, matrix-matched calibrations curves were prepared in order to correct the ionization influence of the sorghum and pearl millet matrixes (Alberts et al., 2019). Coefficients of determination (R2), which indicates the degree of linearity of the respective mycotoxins’ calibration curves, were >0.993 (Figure 5.2).
Mycotoxin concentrations in the FAPAS and Biopure certified quality control samples were within the ranges specified by the supplier for each mycotoxin during each LC-MS/MS run.
Table 5.1 LC-MS/MS conditions for quantification of multiple mycotoxins by positive ESI at capillary voltage 3.5 kV
Analyte Cone
Voltage (V)
Precursor Ion
Quantifier Ion (Collision Energy)
(V)
Qualifier Ion (Collision Energy)
(V)
Aflatoxin B1 50 313 285 (23) 241 (37)
Fumonisin B1 50 722.3 334.3 (40) 352.3 (38)
Fumonisin B2 and B3 50 706.3 318.3 (40) 336.3 (40)
Ochratoxin A 22 404.2 239 (24) 221 (40)
Deoxynivalenol 35 397.1 203.2 (15) 231.2 (12)
Zearalenone 20 319.1 185.0 (23) 187.0 (19)
Nivalenol 15 313.2 175 (25) 295 (8)
Moniliformin 30 97 41 (18) N/A
N/A, not applicable
Table 5.2 Validation parameters of the analytical method for quantification of multiple mycotoxins in sorghum
Analyte Spike level (µg/kg)
Recovery (%)
LOQ (µg/kg)
RSDr(%) Coefficient of determination
(R2)
Aflatoxin B1 10 95 2 1 0.9949
Fumonisin B1 1000 86 5 2 0.9940
Fumonisin B2 500 68 20 4 0.9950
Fumonisin B3 500 76 20 1 0.9958
Ochratoxin A 10 82 20 1 0.9949
Deoxynivalenol 500 86 100 4 0.9975
Zearalenone 200 91 10 3 0.9938
Nivalenol 500 87 10 2 0.9999
Moniliformin 500 85 10 3 0.9959
LOQ, Limit of quantification; RSDr, Relative standard deviation for repeatability; Spike level, supplementation of control grain with a mycotoxin to a specific concentration level
Table 5.3 Validation parameters of the analytical method for quantification of multiple mycotoxins in pearl millet
Analyte Spike level (µg/kg)
Recovery (%)
LOQ (µg/kg)
RSDr(%) Coefficient of determination
(R2)
Aflatoxin B1 10 95 2 2 0.9940
Fumonisin B1 1000 90 2 1 0.9975
Fumonisin B2 500 70 10 3 0.9951
Fumonisin B3 500 80 10 2 0.9963
Ochratoxin A 10 83 10 2 0.9989
Deoxynivalenol 500 85 100 4 0.9980
Zearalenone 200 90 10 2 0.9990
Nivalenol 500 86 10 1 0.9947
Moniliformin 500 85 10 1 0.9943
LOQ, Limit of quantification; RSDr, Relative standard deviation for repeatability; Spike level, supplementation of control grains with a mycotoxin to a specific concentration level
A
m in
5.50 6.00 6.50 7.00 7.50 8.00
%
0 100
F7:MRM of 2 channels ,ES+
TIC HA_CPUT_190509_21 Sm ooth(Mn,1x2)
1.853e+007 FB1
7.29 1094039.1 18522148
B
C
Figure 5.1 LC-MS/MS chromatograms presenting in blue: A, fumonsin B1 (FB1), fumonisin B2
(FB2) and fumonisin B3 (FB3) detected in pearl millet bran sample NAM-17; B, aflatoxin B1 (AFB1) detected in pearl millet malt sample NAM-20, and C, zearalenone (ZEA) detected in sorghum malt sample NAM-24. Fumonisins B1 and B2 were also detected in NAM-24 (chromatograms not shown)
No mycotoxins were detected in any of the raw sorghum and pearl millet samples collected from 10 households of smallholder farmers in Oshakati (Table 5.4). This could mainly be attributed to good agricultural practices. Wood ash added to grains could have effectively limited fungal contamination during storage, by increasing the pH and reducing water activity (aw) (Tangni and Larondelle, 2014). The hot climatic conditions would further assist in reducing aw levels. The grain is stored in airtight plastic containers and woven baskets, which are plastered with clay, thereby reducing aeration and microbial activity. The non-detectible levels of mycotoxins in the raw grain samples do not necessarily imply that mycotoxigenic fungi are absent. Fungal species may be present in low cell numbers or in a dormant state. The combined antimicrobial properties of wood ash and the low aw during storage keeps Fusarium spp. spores dormant. Fusarium spp. are considered field fungi and require an aw of 0.98 to 0.995 for growth (Tangni and Larondelle, 2014).
Processed samples collected at open markets in Oshakati and Ondangwa contained mycotoxins (AF and FB) regulated by the European Union (EU) and the Joint FAO/WHO Expert Committee on Food Additives (JECFA) (ZEA) (Table 5.5). Aspergillus flavus and A.
parasiticus are known to contaminate grains during storage with subsequent production of
carcinogenic aflatoxins (Tangni and Larondelle, 2014). In the current study, 20 and 54% of processed sorghum and pearl millet samples, respectively, contained AFB1 (3-14 µg/kg). 17%
of all bran and malts contained AFB1 at levels above the regulatory maximum level of 5 µg/kg set by the European Commission (Bessaire et al., 2019), with one sample containing 14 µg/kg.
Aflatoxins is classified a Group 1 carcinogen by the Agency for Research on Cancer (IARC) and poses a serious threat to human and animal health by causing hepatotoxicity, teratogenicity, immunotoxicity as well as liver cancer (Pitt, 2012).
FB1 (33%), FB2 (10%) and FB3 (5%) were detected in sorghum and pearl millet bran and malt samples at concentrations below EU regulatory levels of 1000 µg/kg (Table 5.5). 9% of bran and malt samples, however, exceeded the fumonisin regulatory level (200 µg/kg) set by the EC for infants and young children. FB1 is classified a Group 2B carcinogen by IARC and is associated with neural tube defects, stunting in children and oesophageal cancer (Alberts et al., 2019) 4% of processed samples contained ZEA at levels far above recommended EC levels (100 µg/kg) (Ferrigo et al., 2016). JECFA has established a provisional maximum tolerable daily intake (PMTDI) for ZEA of 0.5 µg/kg of body weight (Burger et al., 2014). ZEA is an estrogenic mycotoxin affecting male and female reproductive systems. Contamination by ZEA is mostly caused by F. graminearum, F. equiseti, F. culmorum, F. cerealis and F.
semitectum and ZEA contamination often co-occurs with DON (Ferrigo and., 2016). However, there were non-detectible levels of DON contamination in the processed samples. Co- occurrence of FB1, FB2, FB3 and ZEA was detected in both sorghum and pearl millet malts.
Co-exposure to numerous mycotoxins of young children in particular, is of significant concern (Alberts et al., 2017). Continued exposure to low concentrations of mycotoxins is also a risk factor for human diseases as it is linked to the development of tumours, neural tube defects as well as childhood stunting.
The correlation matrix indicated a strong correlation (R = 0.8-0.83) between levels of F.
verticillioides and F. proliferatum determined with a validated qPCR method (Chapter 4) and FB1, FB2, and FB3 concentrations (Figure 5.3). There was no correlation between the respective Fusarium spp. and DON, ZEA, NIV and MON levels. These results confirm the efficacy of these methods for evaluating specific Fusarium spp. and multiple mycotoxin contamination in grain samples. No correlation was observed between levels of Fusarium spp.
detected with morphological methods and mycotoxin levels (data not shown).
The moisture and temperature conditions during malting provide an ideal environment for fungi to proliferate, and could lead to an exponential increase in mycotoxin concentrations (Agbor Asuk et al., 2020). Contamination may be further enhanced by microflora originating from
home-based malting plants (Noots et al., 1999). The lack of sanitary ware, non-sterilization of equipment and lack of knowledge could result in increased mycotoxin levels. By critically monitoring the grain production process from planting, through to harvesting, processing and marketing, the sources of contamination and critical control points could be identified and managed. Community specific mycotoxin awareness and sustainable education will greatly contribute to reducing mycotoxin contamination during processing of sorghum and pearl millet.
This could involve peer-to-peer training to improve awareness and knowledge, the introduction of community-based mycotoxin reduction methods such as washing of grains and the dissemination of community-specific good agricultural and storage practices (Alberts et al., 2017). Understanding the impact of mycotoxins on human health is critical to further improve the management processes through the development of informed policy strategies and eventually the implementation of regulations. This could ultimately contribute to food safety and security in northern Namibia where communities are exposed to multiple mycotoxins in their staple diet.
Figure 5.2 Matrix-match calibration curves, indicating regression equations and coefficients of determination (R2). A, aflatoxin B1 (AFB1); B, fumonisin B1 (FB1); C, fumonisin B2; D, fumonisin B3; E, ochratoxin A (OTA); F, deoxynivalenol (DON); G, zearalenone (ZEA); H, nivalenol (NIV); I, moniliformin (MON). Solid lines represent actual values. Dashed lines indicate trend lines
Table 5.4 Concentrations (µg/kg) of multiple mycotoxins present in raw sorghum and pearl millet samples collected from households of smallholder farmers in Oshakati
Sample no. Sample description Sample type Sampling
location AFB1 FB1 FB2 FB3 OTA DON ZEA NIV MON
NAM-1S Sorghum Raw grain N1 ND ND ND ND ND ND ND ND ND
NAM-1M Pearl millet Raw grain N1 ND ND ND ND ND ND ND ND ND
NAM-2S Sorghum Raw grain N2 ND ND ND ND ND ND ND ND ND
NAM-2M Pearl millet Raw grain N2 ND ND ND ND ND ND ND ND ND
NAM-3S Sorghum Raw grain N3 ND ND ND ND ND ND ND ND ND
NAM-3M Pearl millet Raw grain N3 ND ND ND ND ND ND ND ND ND
NAM-4S Sorghum Raw grain N4 ND ND ND ND ND ND ND ND ND
NAM-4M Pearl millet Raw grain N4 ND ND ND ND ND ND ND ND ND
NAM-5S Sorghum Raw grain N5 ND ND ND ND ND ND ND ND ND
NAM-5M Pearl millet Raw grain N5 ND ND ND ND ND ND ND ND ND
NAM-6S Sorghum Raw grain N6 ND ND ND ND ND ND ND ND ND
NAM-6M Pearl millet Raw grain N6 ND ND ND ND ND ND ND ND ND
NAM-7S Sorghum Raw grain N7 ND ND ND ND ND ND ND ND ND
NAM-7M Pearl millet Raw grain N7 ND ND ND ND ND ND ND ND ND
NAM-8S Sorghum Raw grain N8 ND ND ND ND ND ND ND ND ND
NAM-8M Pearl millet Raw grain N8 ND ND ND ND ND ND ND ND ND
NAM-9S Sorghum Raw grain N9 ND ND ND ND ND ND ND ND ND
NAM-9M Pearl millet Raw grain N9 ND ND ND ND ND ND ND ND ND
NAM-10S Sorghum Raw grain N10 ND ND ND ND ND ND ND ND ND
NAM-10M Pearl millet Raw grain N10 ND ND ND ND ND ND ND ND ND
Sampling locations (N1-N10) are indicated on the geographical map of Namibia (Chapter 3, Figure 3.1). AFB1, aflatoxin B1; FB1, fumonisin B1; FB2, fumonisin B2; FB3, fumonisin B3; OTA, ochratoxin A; DON, deoxynivalenol; ZEA, zearalenone; NIV, nivalenol; MON, moniliformin; ND, none detected
Table 5.5 Concentrations (µg/kg) of multiple mycotoxins present in processed sorghum and pearl millet samples collected from open markets in Oshakati and Ondangwa
Sample no.
Sample description
Sample type
Sampling
location AFB1 FB1 FB2 FB3 OTA DON ZEA NIV MON
NAM-11 Sorghum Malt OSH M ND 18 ± 0.00 ND ND ND ND ND ND ND
NAM-12 Sorghum Malt OSH M 11 ± 0.88 ND ND ND ND ND ND ND 11
NAM-13 Sorghum Malt OSH M ND 69 ± 0.25 ˂LOQ ND ND ND ND ND ND
NAM-14 Sorghum Malt OSH M ND 15 ± 0.18 ND ND ND ND ND ND ND
NAM-15 Sorghum Malt OSH M ND ND ND ND ND ND ND ND ND
NAM-16 Sorghum Malt OSH M ND ND ND ND ND ND ND ND ND
NAM-17 Pearl millet Bran OSH M ˂LOQ 562 ± 22.65 117 ± 4.97 38 ± 1.31 ND ND ND ND ND
NAM-18 Pearl millet Malt OSH M 6 ± 0.69 ND ND ND ND ND ND ND ND
NAM-19 Pearl millet Malt OSH M 5 ± 0.75 ND ND ND ND ND ND ND ND
NAM-20 Pearl millet Malt OSH M 14 ± 1 ND ND ND ND ND ND ND ND
NAM-21 Pearl millet Malt OSH M 4 ± 0.35 ND ND ND ND ND ND ND ND
NAM-22 Pearl millet Malt OSH M 4 ± 0.35 ND ND ND ND ˂LOQ ND ND ND
NAM-23 Pearl millet Malt OSH M ˂LOQ ND ND ND ND ND ND ND ND
NAM-24 Sorghum Malt ONDW M ND 245 ± 15.56 42 ± 3.77 ˂LOQ ND ND 3184 ± 412.35 ND ND
NAM-25 Sorghum Malt ONDW M 3 ± 0.15 ND ND ND ND ND ND ND ND
NAM-26 Sorghum Malt ONDW M ND 63 ± 0.19 ˂LOQ ˂LOQ ND ND ND ND ND
NAM-27 Sorghum Malt ONDW M ND 73 ± 15.64 ˂LOQ ˂LOQ ND ND 19 ± 7.26 ND ND
NAM-28 Pearl millet Bran ONDW M 3 ± 0.42 ND ND ND ND ND ND ND ND
NAM-29 Pearl millet Malt ONDW M ND ˂LOQ ND ND ND ND ND ND ND
NAM-30 Pearl millet Malt ONDW M ND ND ND ND ND ND ND ND ND
NAM-31 Pearl millet Malt ONDW M ND ˂LOQ ND ND ND ND ND ND ND
NAM-32 Pearl millet Malt ONDW M 4 ± 0.27 ND ND ND ND ˂LOQ ND ND ND
NAM-33 Pearl millet Malt ONDW M ND ND ND ND ND ND ND ND ND
Values represent means ± standard deviations of three replicates. AFB1, aflatoxin B1; FB1, fumonisin B1; FB2, fumonisin B2; FB3, fumonisin B3; OTA, ochratoxin A; DON, deoxynivalenol; ZEA, zearalenone; NIV, nivalenol; MON, moniliformin; OSH M, Oshakati market; ONDW M, Ondangwa market; LOQ, limit of quantification; ND, none detected
Figure 5.3 Correlation matrix indicating correlation coefficients (R) obtained between F.
verticillioides, F. graminearum, F. proliferatum, F. subglutinans, Fumonisin B1 (FB1), fumonisin B2
(FB2), fumonisin B3 (FB3), zearalenone (ZEA), nivalenol (NIV), aflatoxin B1 (AFB1), deoxynivalenol (DON) and moniliformin (MON) levels